MSCs’ conditioned media cytokine and growth factor profiles and their impact on macrophage polarization

MSCs sources

Human BM-MSCs were obtained from the Biobank of the Institute for Regenerative medicine (Sechenov University, Moscow, Russia). Human AT-MSCs, G-MSCs, PL-MSCs, and UC-MSCs were isolated from adipose tissue, gingiva, placenta, and umbilical cord, respectively. In accordance with the Declaration of Helsinki, all the tissue samples were collected after the donor signed an informed consent form approved by the local ethics committee of Sechenov University.

AT-MSCs isolation

For AT-MSCs isolation, the previously reported protocols [23, 24] were adjusted as follows: abdominal subcutaneous adipose tissue biopsy samples were thoroughly washed with Hanks’ solution (BioloT, Russia) supplemented with Diflucan (Pfizer, USA), gentamicin (50 μg/ml, PanEco, Russia), and penicillin/streptomycin (100 U/ml/100 µg/ml, PanEco, Russia). Each sample was minced into small (~ 1 mm3) pieces with scissors and transferred into conical tubes. A fourfold volume of 0.1% collagenase type I solution (PanEco, Russia) in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 supplemented with 2 mM l-glutamine (BioloT, Russia) and 50 mg/ml gentamicin was added to each tube, and enzymatic dissociation was performed for 20 min at 37 °C. Enzymatic digestion was blocked by adding threefold volume of DMEM/F12 supplemented with 2 mM l-glutamine (BioloT, Russia), 10% fetal bovine serum (FBS) (Thermo Fisher, USA), and penicillin/streptomycin (100 U/ml/100 µg/ml, PanEco, Russia). The tubes were then centrifuged at 100g for 10 min at room temperature and the supernatant was discarded. The cell pellet was resuspended in DMEM/F12 supplemented with 2 mM l-glutamine (BioloT, Russia), 10% FBS (Thermo Fisher, USA), and penicillin/streptomycin (100 U/ml/100 µg/ml, PanEco, Russia) and seeded on Petri dishes.

PL-MSCs isolation

PL-MSCs isolation was performed as follows: placenta tissue samples were washed with Hanks’ solution (BioloT, Russia) supplemented with 50 U/ml amphotericin and 100 U/ml penicillin for 12 h. Each sample was minced into small (~ 3 mm3) pieces, and enzymatic dissociation in a 0.15% collagenase I solution (Serva, Germany) was performed at 37 °C for 30 min with constant agitation on Mini Rocker-Shaker (Biosan, Latvia). We then added Hanks’ solution to the suspension, passed it through a 100-μm filter (Becton Dickinson, USA), and centrifuged at 300g for 10 min. The cell pellet was resuspended in DMEM/F12 supplemented with 2 mM l-glutamine (BioloT, Russia), 10% FBS (Hyclone, Germany), 5 mM HEPES (Biomedicals, USA), 100 U/ml penicillin, and 50 µg/ml amphotericin and seeded on Petri dishes.

G-MSCs isolation

G-MSCs were isolated as follows: gingival tissue samples were washed in α-MEM solution (Sigma, USA) supplemented with gentamicin (50 µg/ml, PanEco, Russia) for 12 h. Each sample was minced into ~ 1 mm3 fragments with sterile eye scissors, and enzymatic dissociation in 0.05% collagenase II solution (Sigma, USA) in α-MEM supplemented with 10% FBS (Thermo Fisher, USA) and gentamicin (50 µg/ml, PanEco, Russia) was performed for 12 h at 37 °C. The obtained suspension was centrifuged at 200g for 10 min at room temperature. The cell pellet was resuspended in DMEM/F12 supplemented with 2 mM l-glutamine (BioloT, Russia), 10% FBS (Thermo Fisher, USA), and penicillin/streptomycin (100 U/ml/100 µg/ml, PanEco, Russia) and seeded on Petri dishes.

UC-MSCs isolation

UC-MSCs were isolated as follows: umbilical cord samples were cut into pieces 2 cm long and washed from blood clots in phosphate buffer saline (PBS) supplemented with Diflucan (Pfizer, USA), gentamicin (50 µg/ml, PanEco, Russia), and penicillin/streptomycin (100 U/ml/100 µg/ml, PanEco, Russia). Then, longitudinal incisions were made so that the umbilical vein and umbilical arteries could be removed. Each sample was minced into small (~ 1 mm3) pieces with scissors and transferred into conical tubes. A fourfold volume of 0.2% collagenase NB4 (Serva, Germany), 0.005% hyaluronidase (Sigma, USA), and 10% Accutase (BD BioSciences, USA) in DMEM/F12 medium supplemented with 2 mM l-glutamine (BioloT, Russia) and 50 µg/ml gentamicin (PanEco, Russia) was added to each tube. Enzymatic dissociation was performed at 37 °C for 90 min with constant agitation on Mini Rocker-Shaker (Biosan, Latvia). The obtained suspension was passed through a 70-μm filter (Sigma, USA) and centrifuged at 100g for 7 min at room temperature. The cell pellet was resuspended in DMEM/F12 supplemented with 2 mM l-glutamine (BioloT, Russia), 10% FBS (Thermo Fisher, USA), and penicillin/streptomycin (100 U/ml/100 µg/ml, PanEco, Russia) and seeded on Petri dishes.

Monolayers culturing

The cells were cultured in full growth medium consisting of DMEM/F12 supplemented with 2 mM l-glutamine (BioloT, Russia), 10% FBS (HyClone, USA), insulin–transferrin–sodium selenite (1:100, BioloT, Russia), bFGF (20 ng/ml, ProSpec, Israel), and gentamicin (50 μg/ml, PanEco, Russia) at 37 °C and 5% CO2. MSCs’ morphology and confluence were routinely checked with a phase-contrast microscope Primovert (Carl Zeiss, Germany). The media were replaced every other day, and the cells were passaged at 80% confluence. After 4th passage, the cells were used for spheroid formation and characterization (immunophenotyping and differentiation potential assessment). CM samples were collected on the 3rd day of culturing after 4th passage and stored at − 80 °C until further analysis.

Spheroids formation

Cell spheroids (3000 cells per spheroid) were formed as previously described [25] and cultured likewise. Briefly, agarose non-adhesive microplates created with 3D Petri Dish molds (256 microwells per microplate) (Microtissues, USA) were transferred to the wells of 12-well culture plates, and cell suspension (5.1 × 106 cells/ml) was placed into each microplate. The microplates were incubated for 1 h at 37 °C, 5% CO2 for cell setting, and then covered with full growth medium. CM samples were collected on the 3rd day of culturing and stored at − 80 °C until further analysis.

Immunophenotyping

MSCs monolayers were treated with Versene solution (Invitrogen, USA) and 0.25% trypsin solution (Invitrogen, USA) to obtain single-cell suspensions. Cell spheroids were dissociated prior to the flow cytometry by gentle agitation on Mini Rocker-Shaker (Biosan, Latvia) in 1 ml TrypLE Express (Gibco, USA) per 250 spheroids for 15 min at 37 °C.

The obtained cell suspensions containing at least 1 million cells each were additionally washed in PBS to remove residual culture medium. The pellets were resuspended in PBS containing 1% FBS and aliquoted for subsequent staining with anti-human antibodies for CD105 (conjugated with PerCP-Cy™5.5), CD73 (conjugated with APC), CD90 (conjugated with FITC), CD44 (conjugated with PE), CD19, CD11beta, CD45, CD34, and HLA-DR (all negative markers conjugated with PE) (BD Stemflow™ hMSC Analysis Kit). The staining was performed according to manufacturer`s protocol. Cells stained with isotype control from kit (BD Stemflow™ hMSC Analysis Kit) and unstained cell suspension were used as controls. The antibodies were added in concentrations specified by the manufacturers, and the samples were incubated in the dark for 15 min at room temperature. The samples were then washed with PBS and analyzed on microfluidic cell sorter Sony SH800 (Sony Biotechnology, USA), recording at least 10,000 events per sample. The background level of fluorescence was determined using the unstained cell suspension, while antibody specificity was verified by comparing unstained suspensions with isotype controls. Each marker was subsequently compared to its respective isotype control.

Differentiation potential

The isolated cells’ ability to maintain the multi-lineage differentiation was tested as follows: once 80% confluence was achieved, the medium was changed either to full growth medium (control) or to one of the differentiation media: osteogenic (StemPro™ Osteogenesis Differentiation Kit, ThermoFisher Scientific, USA), chondrogenic (StemPro™ Chondrogenesis Differentiation Kit, ThermoFisher Scientific, USA), or adipogenic (StemPro™ Adipogenesis Differentiation Kit, ThermoFisher Scientific, USA). The cells were cultured for 21 days, the media were changed every 3 days. Following 21 days of differentiation, the cells were washed in PBS and fixed in PFA (4%, pH 6.9, Sigma-Aldrich, Germany) for 20 min at 4 °C.

Osteogenic differentiation was evaluated using Alizarin red staining (Alizarin Red S, Sigma-Aldrich, Germany). The samples were washed in PBS, and a staining solution of 2% Alizarin Red S (pH 4.2) was added to the cells for 30 min.

Chondrogenic differentiation was evaluated using Alcian Blue staining (Sigma-Aldrich, Germany). The samples were washed with 1% acetic acid, then a 1% Alcian Blue staining solution in acetic acid (pH 2.5) was added, and cells were incubated overnight at 4 °C.

Adipogenic differentiation was evaluated using Oil Red O staining (Sigma-Aldrich, Germany). The samples were washed with 60% isopropanol and air-dried for 5 min; then, a 0.2% Oil Red O staining solution in 60% isopropanol was added to the cells for 30 min.

All samples were then thoroughly washed, and images were taken using the Primovert phase-contrast microscope (Carl Zeiss, Germany).

Detection of cytokines and growth factors in MSCs-CM

Cytokines and growth factors in MSCs-CM samples were analyzed with the MILLIPLEX™ MAP Human Cytokine/Chemokine Magnetic Bead Panel (Merck Millipore, USA) utilizing xMAP technology (Luminex, USA). All CM samples contained cytokines and growth factors synthesized by 2.5 × 105 cells per 1 ml.

Briefly, the 96-well plates were washed twice with the buffer provided in the kit. Then, the standards, controls, and samples were added to the wells according to the manufacturer's instructions. All samples were added in duplicate. Control (non-conditioned) culture medium was added as background to the standard and control wells, buffer was added to the sample wells, and ultrasound-pretreated magnetic beads were added to each well. While adding the beads, the tube containing them was vortexed every 30 s to avoid the particles settling and to ensure their even distribution into each well. The plates were then sealed and incubated overnight on Mini Rocker-Shaker (Biosan, Latvia) at 4 °C in the dark. The next day, the plates were washed twice with the buffer using a magnetic washer, so that the beads remained in the wells. The plates were then incubated sequentially with detection antibodies for 1 h and with streptavidin–phycoerythrin for 30 min at room temperature, with constant agitation on Mini Rocker-Shaker (Biosan, Latvia). Then, the plate was washed twice with the buffer on a magnetic washer. Drive fluid provided in the kit was added to each well, the plate was sealed and agitated on Mini Rocker-Shaker (Biosan, Latvia) for 5 min. Then, the plate was analyzed on a MagPix machine (Luminex, USA) with xPONENT (Luminex, USA) software.

Monocyte isolation and differentiation into M0 macrophages

Design of the following experiments including monocyte isolation, differentiation into M0 macrophages, and subsequent treatment with MSCs-CM is graphically presented in Fig. 1.

Fig. 1figure 1

Scheme of the experiments with macrophages. PBMCs were seeded in culture plates and treated with relevant growth factors. On day 6, inducers of M1 or M2 macrophage polarization were added to GM-CSF- or M-CSF-treated macrophages, respectively. UM-MSCs-CM were added into half of the wells (colored pink). On day 8, the markers of M1 or M2 phenotypes were measured

Monocytes were isolated from peripheral blood mononuclear cells (PBMCs). Blood was obtained from healthy volunteers. All donors have signed an informed consent form approved by the local ethics committee of Sechenov University.

Briefly, blood was mixed with PBS and layered onto Histopaque-1077 (Sigma-Aldrich, USA) for density gradient centrifugation at 400g for 40 min. After centrifugation, PBMCs layer was collected, cells were washed twice with PBS and plated at the density of 7.5–8.5 × 105 PBMCs/cm2. Cells were cultured in RPMI complete medium consisting of RPMI-1640 (Corning, USA) supplemented with 10% autologous serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 °C, 5% CO2. After 2 h of incubation, non-adherent cells were washed with PBS, and fresh RPMI complete medium containing 50 ng/ml GM-CSF or 50 ng/ml M-CSF (SCI-store, Russia) was added to adherent monocytes to differentiate them into M0 macrophages for the subsequent experiments with M1 or M2 cells, respectively. The medium was replaced with the fresh one on the 3rd day of culturing.

Treating macrophages with MSCs-CM

On the 6th day of culturing, MSCs-CM were added to both GM-CSF- and M-CSF-treated macrophages in 1:1 ratio with RPMI complete medium (MSC-CM group). Fresh DMEM complete medium was used as a control (Control group). M-CSF-treated macrophages were additionally treated with 20 ng/ml IL-4 (Sigma-Aldrich, USA) to stimulate their M2 polarization (M2 group cultured with M-CSF) and GM-CSF-treated macrophages were additionally treated with 10 ng/ml LPS (Sigma-Aldrich, USA) and 50 ng/ml IFN-γ (Sigma-Aldrich, USA) to stimulate their M1 polarization (Fig. 1). Polarization markers were measured on the 8th day of culturing.

Flow cytometry

On the 8th day of culturing, macrophages were incubated with accutase solution (Sigma-Aldrich, USA) for 5 min at 37 °C, 5% CO2 to obtain single-cell suspensions. The suspensions were added into RPMI complete medium (v/v = 1:8) and centrifuged at 300g for 10 min. After centrifugation, cell pellets were washed twice with PBS to remove residual accutase. Finally, cell pellets were resuspended in PBS containing 1% FBS and aliquoted for subsequent staining with anti-CD-206 antibodies conjugated with PE-Cy7 or with anti-CD86-FITC antibodies (Invitrogen, USA) according to the manufacturer’s protocol. After incubations with antibodies, the samples were washed with PBS and analyzed on microfluidic cell sorter Sony SH800 (Sony Biotechnology, USA) (Additional file 1: Figure S1).

ELISA

On the 8th day of culturing, supernatants were collected from M1 macrophages and centrifuged at 300g for 10 min to exclude cell debris. The concentration of TNF-α, secreted by macrophages polarized toward the M1 state, was measured using an enzyme immunosorbent assay system according to the manufacturer's instructions (“Cytokine”, St. Petersburg, Russia). Optical density was measured using a Multiskan™ FC Microplate Photometer (ThermoFisher, USA).

Statistical data analysis

Python (version 3.10) was used for data analysis. For multiplex analysis, deviations from the expected values of cytokine and growth factor concentrations were calculated as z-scores. Welch’s t test was used to compare differences between two samples. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was applied for multiple comparison. The correlation analysis was conducted by Spearman’s rank test. P values less than 0.05 were considered statistically significant.

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